Objective:To develop specific anti-AML T cells in vitro,and to study their biological characteristics and functions.Methods:Peripheral blood or bone marrow mononuclear cells (MNCs) were isolated from 12 patients with AML,and co-cultured with cytokine combinations in 96 well plates to be induced into dendritic cells (DCs).Cell morphology was observed under an inverted microscope during the first week and immunophenotype was detected by flow cytometry at day 7.Cytokine combination was replaced with high dose IL-2 at day 7 to promote specific anti-AML T cells.T cell phenotype was detected after 4 to 5 weeks.Lactate dehydrogenase (LDH) release assay was used to determine T cell killing activity.Results:After being cultured with cytokine combinations,the typical dendritic appearance with delicate membrane projections was observed.CD80,CD86 and HLA-DR markers were significantly upregulated(P

Background Glial cell line derived neurotrophic factor (GDNF) is determined to have a neurotrophy effect and promoting effect to the growth of axon.GDNF has been applied in ophthalmology.Research showed that artemin,a new member of GDNF family,has a better function in protection of neuron,but seldom relevant document of distruibution of artemin in retina is found so far.Objective The aim of the present study is to investigate the distribution and expression of artemin in normal rat retinal neuron cells and retinal ganglion cells,and imitate diabetic environment to observe the expression of artemin at the condition of high glucose.Methods Retinal tissue was isolated from clean neonatal SD rats and cultured by expand culture method in DMEM/F12 containing 10% fetal bovine serum.40 mmol/L of glucose was added in medium in the seventh day after culture for 12 hours as experimental group.The expression and location of artemin in retina were tested by real-time PCR and cell immunofluorescence assay.Use of experimental animals followed the Management Regulation of experimental animals of Jiangsu Province.Results Cultured cells showed the typical cell body and processes in the seventh day.Cultured retinal ganglion cells (RGCs) presented the red fluorescence for Thy1.1 antibody,and multiple fluorescence label revealed that RGCs exhibited the green fluorescence for artemin antibody and red fluorescence for Thy1.1 antibody,indicating artemin protein was positively expressed in cultured RGCs.The numbers of positive cells for Thy1.1 antibody was (442±9)/high field in normal culture group and (263±7) /high field in 40mmol/L glucose culture group,showing a significant difference between them (P＜0.05).The expression of artemin mRNA in normal culture group and in 40 mmol/L glucose culture group,was showing a considerably difference between them(P＜0.05).Conclusion Artemin can be expressed in cultured retinal neuron cells and RGCs in rats.High glucose environment down-regulate the expression of artemin.This study proved a new idea for protecting RGCs against damage.

Objective To study the effect of baclofen and physical therapy on spasticity after cerebral traumatic injury.Methods20 Patients accepted Baclofen and physical therapy for 3～6 months.Blood and urine routine test,hepatic and renal function were monitored before and every 2 months after treatment.Modified Ashworth scale and Modified Barthel Index(MBI)were used for evaluation.ResultsTheir muscular tension was significantly improved(P<0.01)after treatment,as well as the MBI(P<0.01).1 case was very effective,9 cases were effective,8 cases were improved,2 cases were not effective.No side-effect was observed.ConclusionBaclofen and physical therapy is effective on spasticity after cerebral traumatic injury,especially intervening at the early stage.

Objective To explore the effects of granulocyte colony stimulating factor (G-CSF)on chronic cerebral ischemia in rats,and its possible mechanism.Methods Chronic cerebral ischemia (2-VO) model was prepared and bilateral external jugular veins were isolated.A total of 30 rats were divided into 2 groups at random sham group (received no intervention,n=15) and operative group (received G-CSF or PBS through external jugular vein injection,n=15).At 6 weeks after operation,the rats in operative group were divided into G--CSF group (received G-CSF 10 mg/L,1 ml · kg-1 · d-1,1 times every 24 h for,3 times) and PBS control group (received PBS 10 mg/L,1 ml ·kg 1 · d-11,1 times every 24 h for 3 times).At 8 weeks after the operation,morris water maze was carried out to evaluate the learning and memory ability of the rats.The cell proliferation,threedimensional vascular distribution,ischemic neuronal apoptosis,cell morphological changes in ischemic area and the plasma VEGF levels were detected to explore the possible mechanisms.Results In morris water maze,escape latency at the 2rd to 5th day were significantly lower in G-CSF group than the PBS group (all P＜0.05).The swimming time spent in the first quadrant in G-CSF group was significantly longer than the PBS group (P＜0.05).There was a significant difference in the number of BrdU positive cells in the ischemical area between the G-CSF group and the control group [(27.7±4.76) vs.(10.4 ± 3.7),P =0.030).Three-dimensional quantitative measurements of vascular structure showed that the capillary diameters was smaller in the G-CSF group than in the PBS group [(2.90±0.20) μm vs.(3.45±0.26) μm,P=0.020] and the number of branch points in the boundary regions of ischemia had a significant difference in the G-CSF group compared with the control group [(207.82±10.73) /0.002 mm3 vs.(162.10±9.31) /0.002mm3,P=0.005].Threedimensional cerebral vessel surface area in the ipsilateral hemisphere was increased in the G-CSF group compared with the PBS group [(86498±2896) μm2/0.002 mm3vs.(73976±3826) μm2/0.002 mm3,P=0.003].The number of apoptotic cells in G-CSF group was decreased compared with the PBS group [(32.10±6.70) vs.(56.30±11.20),F=11.89,P=0.043].The electron microscope morphological observations showed inflammatory edema in intercellular gap was significantly reduced in the G-CSF group compared with the PBS group.The level of plasma VEGF was significantly increased in the G-CSF group compared with the PBS group [(58.81±6.61) ng/L vs.(20.81±4.35)ng/L,P=0.025].Conclusions G--CSF can improve the learning and memory ability in the chronic cerebral ischemic rats,and its possible mechanism might involve the nerve protection and the vascular regeneration associated with the VEGF.There is a great prospect for G-CSF in the therapy of chronic cerebral ischemic disease.

·AIM: To investigate whether Erigeron Breviscapus (vant) Hand-Mazz (EBHM) EBHM has neuroprotective effect against N-methyl-D-aspartate (NMDA)-induced neuron death in retinal ganglion cell layer (RGCL).· METHODS: 60 healthy SD rats were randomly divided into four groups. 6 animals were normal control group (group A). The others were divided as group B (EBHM group), group C (normal saline+NMDA group) and group D (EBHM + NMDA group). Each group had 18 rats.10nmol NMDA was intravitreally injected to induce partial damage of the neurons in RGCL in the right eyes of Groups C and D. Same volume PBS was intravitreally injected into the left eyes as self-control. Groups B and D were pre-treated intraperitoneally with 6g/L EBHM solution at a dose of 150mg/kg body weight/day seven days before and after NMDA treatment. Group C were administrated intraperitoneally with 9g/L normal saline at the same time of EBHM injection. Rats were sacrificed at 4,7,14d after NMDA treatment. Flat whole retinas were stained with 5g/L cresyl violet and neuron counting in RGCL from both eyes were observed. Each subgroup had 6 rats.· RESULTS: There was no significant difference of neuron counting in RGCL between the right eye and the left eye in group A (P=0.200). There was no significant difference between normal control group and EBHM group either in the right eyes or in the left eyes at 4, 7 and 14 d respectively after intravitreal injection of 10nmol NMDA in group C and group D. (P=0.636, P=0.193). Neuron counting of RGCL in group C and D was significantly decreased in the NMDA-treated eyes at 4, 7 and 14d after intravitreal injection (P＜0.001). There was no significant difference between self-control eyes group and normai control group(P＞0.05). However, neuron counting was significantly higher in the EBHM+NMDA group than normal saline +NMDA group at 14days after intravitreal injection (P=0.044), but was lowered than normal control group (P＜0.05).· CONCLUSION: EBHM has no effect on neuron counting of RGCL when administered alone in normal rats.The results indicates that EBHM plays a partial protective role in NMDA-induced neuron loss in RGCL in the rats.

Objective To construct the vector that expresses the fusion protein of HIV-Tat protein and red fluorescent protein(mCherry) in mammalian cells,and observe by fluorescence microscopy the intracellular transduction and localization of recombinant protein in cells,in order to obtain a useful tool for the study of the uptake mechanism and intracellular localization of HIV-TAT.Methods With the designed primer coding mCherry sequence,the mCherry gene was amplified by PCR with the vector pmCherry-C2 as template,and inserted into vector pET14b-His-TAT to construct the expression vector pET14b-His-TAT-mCherry.The constructed vector was then transformed into E.coli BL21(DE3),which had been identified by PCR and double digested with restriction endonuclease,followed by sequencing.After IPTG induction,the recombinant protein of His-TAT-mCherry was lyzed and analyzed with SDS-PAGE.Purified His-TAT-mCherry recombinant protein was added to Hela cells and the fluorescence was observed to evaluate the transduction efficiency.Results The results of identification by PCR,digestion with restriction endonuclease and sequencing indicated that the vector His-TAT-mCherry was correctly constructed.His-TAT-mCherry fusion protein was expressed in mammalian Hela cell line and purified successfully,and the fusion protein showed cellular transduction activity.It was found by fluorescence microscopy that the red fluorescence protein located mainly over the cytoplasm,and also the membrane to some extent.Conclusion The expression vector is successfully constructed for HIV-TAT labeled with mCherry sequence.Effective expression and purification of this fusion protein is achieved.It has been observed that the constructed vector may be expressed in mammalian Hela cell under active condition.Thus,it might be useful in the study of uptake mechanism and intracellular localization of HIV-TAT.

Objective To study the curative effect of noninvasive ventilation on the patients of acute serious bronchial asthma complicated with pneumothorax. Methods Forty patients were randomly assigned to receive BiPAP ventilation immediately after thoracic closing drainage, or nasal catheter oxygen inhalation after thoracic closing drainage. Results After 24-hour ventilation, the levels of PO_ 2, SO_ 2, PCO_ 2 and breath rate between two groups were of remarkable difference (P0.05). The level of PO_ 2, SO_ 2, PCO_ 2, and heart rate, respiratory rate of the BiPAP patients were of remarkable difference (P

AIM To observe the effects of Wuwei Wentong Chubi Capsules (Cinnamomi Ramulus,Poria,Epimedii Folium,etc.) on autophagy proteins of Beclin-1 and LC3-Ⅱ in adjuvant-induced arthritis rats and to explore the possible mechanism of action.METHODS Sixty SD rats were randomized into six groups:normal group,model group,Wuwei Wentong Chubi Capsules (0.8,1.6,3.2 g/kg) groups and tripterygium glycosides (TPT,40 mg/kg) group.In addition to the normal group,adjuvant arthritis (AA) was induced with Freund's complete adjuvant.From the 2th day after injection of FCA,Wuwei Wentong Chubi Capsules with different doses and TPT were given by gavage once a day for 12 days.At the end of the experiment,ankle-joint samples were taken to examine the degree of AA by HE.Beclin-1 and LC3-Ⅱ mRNA were determined by real-time fluorescent quantitative PCR.Meanwhile,the protein expression levels of Beclin-1 and LC3-Ⅱ were determined by immunofluorescence histochemical staining and Western blot.RESULTS As compared with the model group,Wuwei Wentong Chubi Capsules (1.60,3.20 g/kg) not only significantly reduced histopathological injuries,but also effectively up-regulated mRNA and protein expressions of Beclin-1 and LC3-Ⅱ.CONCLUSION Wuwei Wentong Chubi Capsules has a therapeutic action on AA in rats,which might be partly associated with promoting autophagy,decreasing excessive proliferation of synovial cells,leading to the reduction of damage to articular cartilage.

Medics play a very important role in military medical supporting forces who take on lots of basic work of medical units.It was assigned to us to train the medics for the first time this year in our university.To enhance the teaching effect of the course named first aid in battlefield,a series of measures were taken including modularizing the teaching contents,diversifying the teaching means,dividing students into some groups,strengthening the practice and examinations based on the traits of the medics and so on.At last,some countermeasures were put forward aiming at the process of teaching.

Objective To evaluate the safety and efficacy of endoscopic ultrasound (EUS) guided ethanol ablation of benign insulinoma and compare its' advantages and disadvantages with surgical treatment. Methods From April 2011 to February 2016, clinical data of 38 patients with benign insulinoma treated by EUS-guided ethanol ablation or surgical treatment were retrospectively analyzed. Results 97.4% (37/38) patients had a typical clinical manifestation of Whipple's triad, and the I/G ratio of 82.9% patients (29/35) was more than 0.3 with their onset of hypoglycemia. The positive preoperative etiologic diagnosis rates of transabdominal ultrasonography, CT, MRI, PET/CT and EUS were 50.0%, 67.6%, 66.7%, 75.0%, 89.7% respectively. In the current study, 18 patients underwent EUS-guided ethanol ablation (EUS-FNI group) and 20 patients received surgicaltreatment (surgical group). Compared with the surgical group, the operation time, intraoperative hemorrhage volume, postoperative complications, length of stay and hospitalization costs were significantly reduced in the EUS-FNI group (P < 0.05). No treatment-related complications was observed in EUS-FNI group, while 40.0% (8/20) patients in surgical group had complications. During the follow-up period, all these patients maintained stable blood glucose without taking medication, and there's no recurrence of insulinoma in EUS-FNI group after the last treatment with alcohol injection; In surgical group, only 90.0% (18/20) patients had no recurrence, episode of hypoglycemia was less after the operation in 10.0% (2/20) patients. Conclusion EUS-guided ethanol ablation of benign insulinoma is safe and effective, compared with traditional surgical treatment, EUS-guided ethanol ablation is minimally invasive, costs less, recovers fast after treatment and has fewer complications.